Ischemic stroke is a major cause of morbidity and mortality in the western world. Despite intensive research in this field, current options for treatment are still limited. One approach to preventing stroke or promoting recovery after stroke is to encourage the growth of new blood vessels (angiogenesis), to restore vascular delivery to the ischemic area. Angiogenesis is regulated by growth factors, cytokines, and extracellular matrix (ECM) proteins like fibronectin. The hypothesis to be tested by this proposal is that cerebral ischemia induces brain endothelial cell expression of the a5?1 and av?3 integrins, and that fibronectin promotes cerebral angiogenesis, via these integrins. Studies outside the central nervous system (CNS) show that the a5?1 and av?3 integrins are strongly induced on sprouting blood vessels, and functional blockade of these integrins inhibits angiogenesis in animal models of tumor formation and diabetic neovascularization. Evidence from our laboratory suggests that these integrins may also promote angiogenesis in the CNS: (i) developmental angiogenesis is strongly associated with brain endothelial expression of the a5?1 integrin, (ii) the a5?1 integrin is also strongly induced on angiogenic endothelial cells during hypoxia in the adult CNS, (iii) following cerebral ischemia, ?1 integrin mRNA levels are increased in the ischemic penumbra, where new vessel sprouting occurs, (iv) the av?3 integrin is induced on angiogenic endothelial cells rapidly following cerebral ischemia, and (v) in vitro studies of brain endothelial cells demonstrate that the survival and mitogenic effects of fibronectin are mediated by both the a5?1 and av?3 integrins. The hypothesis will be tested using a combination of in vivo and in vitro approaches. Endothelial expression of the a5?1 and av?3 integrins will be examined in mouse models of focal cerebral ischemia, and cerebral hypoxia. The role of the av?3 integrin in cerebral angiogenesis will be directly examined in both animal models using ?3 integrin null mice. Potential roles for the a5?1 and av?3 integrins in regulating mouse brain endothelial cell behavior will be addressed by using endothelial cells deficient in these integrins. Finally, the time-course and cellular source of the pro-angiogenic factor, TGF-?1 will be defined in these systems. This study has three Specific Aims: 1) demonstrate induction of the a5?1 and av?3 integrins on brain endothelial cells following ischemia or hypoxia in cerebral tissue;2) demonstrate an instructive role for the a5?1 and av?3 integrins in promoting brain endothelial cell survival, proliferation, migration, and capillary formation;and 3) define the time-course and cellular origin of the pro-angiogenic factor, TGF- ?1 following cerebral ischemia and cerebral hypoxia. These studies will directly test the idea that the a5?1 and av?3 integrins play an important role in promoting cerebral angiogenesis in response to ischemia. Based on this work, manipulation of the expression level or signaling of these integrins are likely to provide a promising approach to enhance cerebral angiogenesis, either before or immediately after ischemic stroke.

Public Health Relevance

The overall aim of this Project is to examine the notion that cerebral ischemia induces endothelial cell expression of the a5?1 and av?3 integrins, and that fibronectin promotes new blood vessel formation in the brain via these integrins. Based on this work, manipulation of the expression levels or signaling pathways of these integrins may provide a promising therapeutic approach to enhance cerebral blood vessel growth, either before or immediately after ischemic stroke. If successful, this work could have major impact on the incidence and mortality of ischemic stroke.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS060770-05
Application #
8286351
Study Section
Brain Injury and Neurovascular Pathologies Study Section (BINP)
Program Officer
Bosetti, Francesca
Project Start
2008-07-15
Project End
2013-06-30
Budget Start
2012-07-01
Budget End
2013-06-30
Support Year
5
Fiscal Year
2012
Total Cost
$406,241
Indirect Cost
$191,866
Name
Scripps Research Institute
Department
Type
DUNS #
781613492
City
La Jolla
State
CA
Country
United States
Zip Code
92037
Welser-Alves, Jennifer V; Boroujerdi, Amin; Tigges, Ulrich et al. (2014) Analysis of TNF-alpha-mediated cerebral pericyte remodeling. Methods Mol Biol 1155:81-93
Boroujerdi, Amin; Tigges, Ulrich; Welser-Alves, Jennifer V et al. (2014) Isolation and culture of primary pericytes from mouse brain. Methods Mol Biol 1135:383-92
Welser-Alves, Jennifer V; Boroujerdi, Amin; Milner, Richard (2014) Isolation and culture of primary mouse brain endothelial cells. Methods Mol Biol 1135:345-56
Tigges, Ulrich; Boroujerdi, Amin; Welser-Alves, Jennifer V et al. (2013) TNF-? promotes cerebral pericyte remodeling in vitro, via a switch from ?1 to ?2 integrins. J Neuroinflammation 10:33
Welser-Alves, Jennifer V; Milner, Richard (2013) Microglia are the major source of TNF-* and TGF-*1 in postnatal glial cultures; regulation by cytokines, lipopolysaccharide, and vitronectin. Neurochem Int 63:47-53
Welser-Alves, Jennifer V; Boroujerdi, Amin; Tigges, Ulrich et al. (2013) Endothelial ?4 integrin is predominantly expressed in arterioles, where it promotes vascular remodeling in the hypoxic brain. Arterioscler Thromb Vasc Biol 33:943-53
Welser, Jennifer V; Milner, Richard (2012) Derivation of microglia-free astrocyte cultures from neural stem cells. Methods Mol Biol 814:81-91
Li, Longxuan; Liu, Fudong; Welser-Alves, Jennifer V et al. (2012) Upregulation of fibronectin and the ?5?1 and ?v?3 integrins on blood vessels within the cerebral ischemic penumbra. Exp Neurol 233:283-91
Welser, Jennifer V; Milner, Richard (2012) Use of astrocyte-microglial cocultures to examine the regulatory influence of astrocytes on microglial activation. Methods Mol Biol 814:367-80
Welser-Alves, Jennifer V; Crocker, Stephen J; Milner, Richard (2011) A dual role for microglia in promoting tissue inhibitor of metalloproteinase (TIMP) expression in glial cells in response to neuroinflammatory stimuli. J Neuroinflammation 8:61

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